Analytical and Bioanalytical Chemistry Electronic Supplementary Material Determination of the protein content of complex samples by aromatic amino acid analysis, liquid chromatography-UV absorbance, and colorimetry

Fast and accurate determination of the protein content of a sample is an important and non-trivial task of many biochemical, biomedical, food chemical, pharmaceutical, and environmental research activities. Different methods of total protein determination are used for a wide range of proteins with highly variable properties in complex matrices. These methods usually work reasonably well for proteins under controlled conditions, but the results for non-standard and complex samples are often questionable. Here, we compare new and well-established methods, including traditional amino acid analysis (AAA), aromatic amino acid analysis (AAAA) based on the amino acids phenylalanine and tyrosine, reversed-phase liquid chromatography of intact proteins with UV absorbance measurements at 220 and 280 nm (LC-220, LC-280), and colorimetric assays like Coomassie Blue G-250 dye-binding assay (Bradford) and bicinchoninic acid (BCA) assay. We investigated different samples, including proteins with challenging properties, chemical modifications, mixtures, and complex matrices like air particulate matter and pollen extracts. All methods yielded accurate and precise results for the protein and matrix used for calibration. AAA, AAAA with fluorescence detection, and the LC-220 method yielded robust results even under more challenging conditions (variable analytes and matrices). These methods turned out to be well-suited for reliable determination of the protein content in a wide range of samples, such as air particulate matter and pollen. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-022-03910-1.


Protein characterization
To characterize the six test proteins, SDS-PAGE and MALDI-TOF MS measurements were performed ( Figure S6, S7).
A high-purity, low endotoxin, low IgG, monomeric bovine serum albumin (BSA) crystallized powder preparation with near native albumin characteristics was used. An aqueous solution of this BSA is termed high-purity BSA thereafter and was used as a secondary reference (1.07 mg/mL determined by AAAA(Phe)). The expected mass is 66.4 kDa, which was confirmed by MALDI-TOF MS measurements, and also the SDS-PAGE showed a single protein band at ∼ 65 kDa. The integrated LC-220 chromatogram gave a purity result of 98.8 % (related to the UV signal). Of note is that the LC-220 protein purity does not account for any inorganic or other non-absorbing material.
Chicken avidin forms a strong non-covalent complex with biotin, and is often used in biochemical assays such as ELISA or western blot [1]. Avidin is a basic protein with an isoelectric point of 10.0-10.5, and approx. 10 % of its total mass results from carbohydrates. The expected mass from the protein sequence is 13.9 kDa, and MALDI-TOF MS gave a mass of 15.7 kDa. SDS-PAGE showed one protein band at ∼ 17 kDa. The integrated LC-220 chromatogram gave a purity result of 87 %.
Myoglobin is an essential hemoprotein in striated muscle [2]. Myoglobin is a single chain heme protein with no disulfide b ridges o r f ree S H-groups a nd a n i ron c ontent of 0.25-0.32 % (information of the manufacturer). The expected mass is 16.9 kDa, which was confirmed by MALDI-TOF MS measurements; the SDS-PAGE showed one protein band at ∼ 16 kDa. The integrated LC-220 chromatogram gave a purity result of 96.6 %.
Jacalin, a galactose-binding lectin from jackfruit seeds is able to bind to O-linked glycoproteins, particularly human IgA, which makes it useful for isolating plasma glycoproteins, investigating IgA nephropathies, and tumor detection [3]. The expected mass is 16.2 kDa, which was confirmed by M ALDI-TOF M S. T he S DS-PAGE showed two protein bands between ∼ 15-17 kDa. The integrated LC-220 chromatogram gave a purity result of 82.3 %.
Transferrins are iron-transport proteins [4]. The iron-deficient t ransferrin i s called apotransferrin. We used bovine apotransferrin (Apo) in form of a sterile filtered lyophilized sample with a purity of 95 % and an iron content < 40 ppm (information of the manufacturer). The expected mass is 75.8 kDa, and the measured MALDI-TOF MS gave a mass of 77.6 kDa. The SDS-PAGE showed two protein bands between ∼ 50-80 kDa. The integrated LC-220 chromatograms gave a purity result of 96.5 %.
Protein G is a single non-glycosylated protein, which can bind to a broad range of mouse and human IgG subclasses. In our study, the recombinant form of Protein G is used [5]. The expected mass of recombinant protein G (rPG) is 21.8 kDa, which was confirmed b y M ALDI-TOF M S m easurements, a nd t he S DS-PAGE s howed several protein bands between ∼ 28-35 kDa. The integrated LC-220 chromatogram gave a purity result of 99.1 %.
According to our quality evaluation of the investigated proteins (SDS-PAGE, MALDI-TOF MS, LC-220), aliquots of the same aqueous high-purity BSA solution were used as our calibration solution with a concentration of 1.07 ± 0.03 mg/mL determined by AAAA(Phe). If not stated differently, all method calibrations were done with this high-purity BSA solution.